Objective To investigate the protective effects and mechanisms of hawthorn total flavonoids against myocardial ischemia-reperfusion (I/ R) injury in rats via the HIF-1 signaling pathway. Methods An ex vivo rat heart I/ R model was established. Animals were randomized into five groups: Sham, I/ R, low-dose hawthorn total flavonoids (HTF-L), high-dose hawthorn total flavonoids (HTF-H), and HTF-H combined with the PI3K inhibitor LY294002(HTF-H+LY). Cardiac function (LVSP, LVEDP, and ±dp / dt_max), myocardial apoptosis (TUNEL),oxidative stress markers (SOD and MDA), and protein expression of HIF-1α and upstream pathways (PI3K/ Akt and MAPK) were assessed after 60 min of reperfusion. Results Compared with the Sham group, the I/ R group exhibited significantly decreased cardiac function ( LVSP and ± dp / dt _ max, both P<0. 01), and increased apoptosis and oxidative stress levels (both P<0. 01). HTF treatment significantly improved cardiac function (P<0. 05 or P<0. 01),suppressed apoptosis and oxidative stress (P<0. 05 or P<0. 01) in a dose-dependent manner. Western blot showed that HTF significantly upregulated HIF-1α and upstream PI3K/ Akt / MAPK signaling pathway proteins (P<0. 05 or P<0. 01). PI3K inhibition partially reversed the protective effects of HTF (P<0. 05). Conclusions Hawthorn total flavonoids can alleviate myocardial injury induced by ischemia / reperfusion (I/ R) via activation of the HIF-1 signaling pathway; the underlying mechanism involves inhibition of cardiomyocyte apoptosis and oxidative stress. These findings provide experimental evidence for both the application of hawthorn as a food-medicine homologous agent in the prevention and treatment of cardiovascular diseases and for research into its molecular mechanisms.